Telomere maintenance assays

Abstract

The strand of telomeric DNA that runs 5' to 3' towards a chromosome end is typically G-rich. Due to the properties of conventional DNA polymerases, telomerase-generated tails of this G-rich strand are expected on only one end of individual linear DNA molecules. In Saccharomyces, TG1-3 tails are detected on chromosome and linear plasmid telomeres late in S-phase. Moreover, the telomeres of linear plasmids can interact when the TG1-3 tails are present. Molecules were generated in vitro that mimic the structures predicted for telomere replication intermediates. These in vitro generated molecules formed telomere-telomere interactions similar to those on molecules isolated from yeast but only if both ends that interacted had a TG1-3 tail. Moreover, TG1-3 tails were generated in vivo in cells lacking telomerase. These data suggest a new step in telomere maintenance cell cycle regulated degradation of the C1-3A strand which can generate a potential substrate for telomerase and telomere binding proteins at every telomere.

What is claimed is: 
     
       1. A method for detecting 5'-3' exonuclease activity in a sample comprising the steps of: contacting the sample with a substrate linear double-stranded nucleic acid molecule wherein at least one end of such molecule does not have a 3' overhang; and   determining whether a resulting linear nucleic acid molecule has 3' overhangs at both ends, whereby the presence of 3' overhangs at both ends of the resulting nucleic acid molecule indicates the presence of 5'-3' exonuclease activity in the sample.   
     
     
       2. The method of claim 1 wherein the sample comprises a cell transfected with the linear nucleic acid molecule. 
     
     
       3. The method of claim 2 wherein the cell is a yeast. 
     
     
       4. The method of claim 2 wherein the cell is mammalian cell. 
     
     
       5. The method of claim 2 wherein the 3' overhang on the first end comprised TG 1-3  repeats and the cell is a yeast cell or a mammalian cell. 
     
     
       6. The method of claim 2 wherein the cell lacks telomerase activity and the cell is a yeast cell or a mammalian cell. 
     
     
       7. The method of claim 1 wherein the 5'-3' exonuclease is FEN-1 or RAD27. 
     
     
       8. The method of claim 1 wherein the 5'-3' exonuclease is a mammalian homologue of FEN-1 or RAD27. 
     
     
       9. The method of claim 1 wherein the 5'-3' exonuclease is a human homologue of FEN-1 or RAD27. 
     
     
       10. The method according to claim 1, wherein the sample contains a known quantity of a 5'-3' exonuclease and the method additionally comprises the steps of: determining the amount of 5'-3' exonuclease activity in the sample after contact with a compound; and comparing the amount of the 5'-3' exonuclease activity in the sample with a standard activity for the same quantity of the exonuclease, whereby a difference between the amount of activity in the sample and the standard activity indicates that the compound modulates the activity of the 5'-3' exonuclease.   
     
     
       11. The method according to claim 10, wherein the sample contains an enzyme which regulates a 5'-3' exonuclease, and the method additionally comprises the steps of: comparing the amount of 5'-3' exonuclease activity in the sample before and after contact with a compound, whereby a difference between the amount of activity in the sample before and after contact with the compound, beyond that which can be attributed to the effect of the compound directly on the exonuclease, indicates that the compound modulates the activity of the enzyme.   
     
     
       12. A method for detecting 5'-3' exonuclease activity in a sample comprising the steps of: contacting the sample with a substrate linear double-stranded nucleic acid molecule, wherein at least one end of such molecule does not have a 3' overhang; and   determining whether a resulting nucleic acid molecule has 3' overhangs at both ends based on whether the resulting nucleic acid molecule undergoes end-to-end interactions,   whereby the presence of 3' overhangs at both ends of the resulting linear nucleic acid molecule indicates the presence of 5'-3' exonuclease activity in the sample.   
     
     
       13. The method of claim 12 wherein one end of the substrate linear nucleic acid molecule has a 3' overhang and the other end has a blunt end. 
     
     
       14. The method of claim 12 wherein the resulting linear nucleic acid molecule is determined to undergo end-to-end interactions if the resulting molecule circularizes. 
     
     
       15. The method of claim 12 wherein the resulting linear nucleic acid molecule is determined to undergo end-to-end interactions if the resulting molecule forms linear concatenations of at least three linear nucleic acid molecules. 
     
     
       16. The method of claim 15 wherein the step of determining whether both ends of the linear nucleic acid molecule have 3' overhangs comprises determining the length of the 3' overhangs in the linear nucleic acid molecule. 
     
     
       17. The method as in any of claims 1-16 wherein the 5'-3' exonuclease is involved in telomerase maintenance.